Deposition of amyloid in pancreatic islets is a common feature in human Type II diabetic patients. The major protein forming these amyloid particles, called amylin, has a propensity to form fibril amyloid structures. Proceedings of the National Academy of Sciences USA 84(23):8628-32, 1987. Amylin is a 37 amino acid protein which, in its fully active form, is carboxy-amidated and has a disulfide bridge between the cysteine residues found at positions 2 and 7. Amylin plays a role in control of systemic concentrations of glucose, and has been proposed as a useful therapeutic agent. See, e.g., Leighton and Cooper, 15 TIBS 295, 1990. Human amylin is described and claimed in U.S. Pat. No. 5,367,052, entitled “Amylin Peptides,” and U.S. Pat. No. 5,124,314, entitled “Pharmaceutical Compositions Containing Amylin.” Amylin has been reviewed in the literature, for example, in Gaeta, L. S. L. and Rink, T. J., 3 Med. Chem. Res. 483-490, 1994, Pittner, R. A. et al., 55S J. Cell. Biochem. 19-28, 1994, and Rink, T. J. et al., 14 TIPS 113-118, 1993.
Therapeutic opportunities for insulin-using and other people with diabetes who are deficient in amylin or for whom amylin therapy would be of benefit, and hormone blockage for other people, for example, the obese and Type II diabetics and those with insulin resistance who may have elevated plasma amylin or undesired amylin activity have been pursued. The use of amylin agonists, including amylin itself, for the treatment of diabetes is described and claimed in U.S. Pat. No. 5,175,145. The use of amylin antagonists for the treatment of Type II diabetes mellitus, obesity and essential hypertension, and insulin resistance, are described and claimed in U.S. Pat. Nos. 5,266,561, 5,280,014, 5,281,581, and 5,364,841.
The most severe form of the disease is Type I (juvenile-onset) diabetes. There are an estimated 1 million Type I diabetics in the U.S. who need daily insulin injections for survival. Their quality of life is often markedly affected by the rigors of their daily metabolic imbalances, in particular hypoglycemic attacks (dangerously low blood glucose) and by the onset of serious long-term complications, including blindness, kidney failure, impotence, ulcers, amputations and atherosclerosis (NIH Diabetes Complications and Control Trial).
Type II (adult-onset) diabetes afflicts over 10 million Americans, who are also subject to the same complications. Impaired glucose tolerance, a risk factor for Type II diabetes and cardiovascular disease, is thought to affect another 20 million people in the U.S. and is not treatable by any known regimen. There is also an alarming increase in the incidence of Type II diabetics in groups of populations around the world, whose standard of living is increasing through economic development or migration. Sulfonylureas are the primary oral antihyperglycemic diabetic medications sold in the U.S. Discovered in the 1940's, these compounds do not address the underlying causes of Type II diabetes and, in many cases, are not effective or lose their efficacy after a few years of treatment. Type II diabetics do not lack insulin, rather they are insulin resistant, so that insulin does not work properly and the insulin secretory responses are disordered.
After a meal, the pancreas secretes insulin in response to a rise in glucose. Insulin stimulates the uptake of glucose into muscle and fat, and signals the liver to reduce glucose production; this results in a return of blood glucose to normal levels. In muscle, large amounts of glucose are stored as glycogen. Some of the glycogen is broken down into lactate, which circulates to the liver and can be converted back into glucose and stored as glycogen. Between meals the liver breaks down these glycogen stores to provide glucose to the brain and other tissues. This cycle in which glycogen is effectively transferred from muscle to liver is known as the Cori cycle. The stimulus for this flux from muscle to liver under resting conditions remain unidentified; recent results indicate that amylin provides a major stimulus to this pathway.
Amylin has been demonstrated to have direct metabolic effects in both skeletal muscle and the pancreas. In skeletal muscle, amylin acts as a non-competitive antagonist of insulin, reducing insulin-stimulated incorporation of glucose into glycogen. In vitro studies indicate that amylin reduces glycogen synthese activity and favors the formation of an active form of glycogen phosphorylase, the enzyme that converts glycogen into glucose 6-phosphate. The actions of amylin on skeletal muscle promote glycogen breakdown, thus stimulating lactate formation and increasing turnover of the Cori cycle. Amylin is co-secreted with insulin from pancreatic beta cells and has been demonstrated to suppress insulin secretion. It appears to provide feedback regulation of the beta-cell, in order to modulate insulin secretory activity.
It is believed that amylin plays a role in the regulation of glucose uptake from ingested food into blood, and that amylin or amylin agonist therapy in diabetics, particularly insulin-using diabetics, such as Type I diabetics and late-stage Type II diabetics, will smooth the excessive glucose rises that these patients typically experience after meals. Deficiency of an important hormone such as amylin which has been reported to have effects on carbohydrate, fat and bone metabolism, may also disrupt other important physiological mechanisms. The co-administration of amylin, or an amylin agonist which exerts the physiological effects of amylin, will significantly improve existing insulin therapy by restoring the appropriate metabolic balance.
Many factors affect the stability of a pharmaceutical product, including the chemical reactivity of the active ingredient(s), the potential interaction between active and inactive ingredients, the manufacturing process, the dosage form, the container-closure system, and the environmental conditions encountered during shipment, storage, handling and length of time between manufacture and usage. Pharmaceutical product stability is determined by the chemical stability as well as the physical stability of the formulation. Physical factors including heat and light may initiate or accelerate chemical reactions.
Optimal physical stability of a formulation is very important for at least three primary reasons. First, a pharmaceutical product must appear fresh, elegant and professional, when it is administered to a patient. Any changes in physical appearance such as color changes or haziness can cause a patient or consumer to lose confidence in the product. Second, because some products are dispensed in multiple-dose containers, uniformity of dose content of the active ingredient over time must be assured. A cloudy solution or a broken emulsion can lead to a non-uniform dosage pattern. Third, the active ingredient must be available to the patient throughout the expected shelf life of the preparation. A breakdown of the product to inactive or otherwise undesired forms can lead to non-availability of the medicament to the patient.
Stability of a pharmaceutical product, then, may be defined as the capability of a particular formulation to remain within its physical, chemical, microbiological, therapeutic and toxicological specifications. A stable solution retains its original clarity, color and odor throughout its shelf life. Retention of clarity of a solution is a main concern in maintaining physical stability. Solutions should remain clear over a relatively wide temperature range such as about 4° C. to about 37° C. At the lower range an ingredient may precipitate due to a lower solubility at that temperature, while at higher temperatures homogeneity may be destroyed by extractables from the glass containers or rubber closures. Thus, solutions of active pharmaceutical ingredients must be able to handle cycling temperature conditions. Similarly, a formulation should retain its color throughout this temperature range, and its odor should be stably maintained.
Small peptides are typically unstable and are susceptible to degradation in aqueous solution. In this regard, once a human amylin agonist or amylin has less than approximately 90% of its labeled potency, it is no longer considered to be suitable for administration to a patient. Various types of molecules such as sugars, surfactant, amino acids and fatty acids, used singly or in combination, have been used in efforts to stabilize protein and peptide products against degradation. See Wang and Hanson, J. Parenteral Science and Technology Supplement, 1988, Technical Report No. 10 (describing parenteral formulations of proteins and peptides); Manning et al., 6 Pharmaceutical Research, 1989. Examples of excipients such as buffers, preservatives, isotonic agents, and surfactants are also known in the art. See 21 C.F.R. §180.22 et seq. (defining recognized food additives); Wang and Kowal, 34 J. Parenteral Drug Association 452, 1980 (describing various excipients); A. R. Gennaro et al., 17th Remington's “Pharmaceutical Sciences,” 1985; Avis et al., Pharmaceutical Dosage Forms: Parenteral Medications, Vol. 1, 1992, all of which, including the definitions of various useful excipients, are hereby incorporated by reference herein.